This project focuses on glycoconjugates of Schwann cells and oligodendrocytes during myelination and demyelination. A major aspect of the research concerns the myelin-associated glycoprotein (MAG), which is localized in periaxonal glial membranes of myelinated fibers and functions in transmitting signals between axons and myelin-forming cells. MAG is in the "siglec" subgroup of the immunoglobulin superfamily and binds to glycoconjugates containing terminal alpha2-3-linked sialic acid. This suggests that its axonal receptor or ligand could be a glycoprotein or ganglioside. Furthermore, the sialic acid moieties on other axonal or glial glycoconjugates and on MAG itself could modulate its function, because it is known that the function of other siglec family members are regulated by the expression of cis and trans sialic acid moieties. Expression of alpha 2-3 sialic acid on MAG and other glycoproteins of nerve is increased in some inherited mouse and human neuropathies, and our results suggest that this contributes to pathology by interfering with MAG-mediated signaling. However, the identity of the functional binding-partner(s) for MAG is not known. Our experiments involving Western blot overlay and co-immunoprecipitation demonstrated that MAG binds to a phosphorylated isoform of microtubule-associated protein 1B (MAP1B) expressed in dorsal root ganglion neurons (DRGNs) and axolemma-enriched fractions from myelinated axons of brain, but not to the isoform of MAP1B expressed by glial cells. These results, plus our previous demonstration that some MAP1B is expressed as a neuronal plasma membrane glycoprotein, is consistent with its being a binding-partner for MAG on the axonal surface. Binding sites for a MAG-Fc chimera on DRGNs co-localized with MAP1B on neuronal varicosities, and MAG and MAP1B also co-localized in the periaxonal region of myelinated axons. In addition, expression of the phosphorylated isoform of MAP1B was significantly increased when DRGNs were co-cultured with MAG-transfected COS cells. Based on these findings, we hypothesize that a MAG/MAP1B interaction could provide a structural link between the periaxonal membrane of the myelin-forming cell and the axonal cytoskeleton, thereby contributing to the known capacity of myelin to affect the structure and stability of myelinated axons (see below). MAG-null mice myelinate relatively normally during early development, but neuropathological changes occur as the mice age. Biochemical and morphological studies on MAG-null mice over one-year old were undertaken to characterize these pathological changes in greater detail. As reported last year, the results on the CNS were indicative of oligodendroglial pathology and consistent with the morphological demonstration of a "dying back" oligodendrogliopathy, similar to that occurring in some demyelinating lesions in multiple sclerosis. However, the pathology in the PNS is quite different, being characterized by axonal degeneration in association with a reduction of axonal caliber caused by cytoskeletal abnormalities including decreased expression and phosphorylation of neurofilaments. We have shown that this decreased phosphorylation of neurofilaments in MAG-null mice is due in part to decreased activities of extracellular signal regulated kinases (ERKs) and cyclin dependent kinase 5 (cdk5). Although previous studies had not revealed electrophysiological abnormalities in younger MAG-null mice, our electrophysiological evaluation of these one year-old mutants showed mild reductions in conduction velocity and compound muscle action potential amplitudes. These findings are indicative of an axonopathy resembling type 2 Charcot-Marie-Tooth disease in humans and demonstrate that mutation of a Schwann cell protein can cause functionally significant axonal degeneration. The explanation could be either that MAG itself is part of a signal transduction pathway that is necessary for axonal maintenance or that there is a general breakdown of the Schwann cell-axon junction in the absence of MAG that disrupts signaling to the axon by other molecules. The direct involvement of MAG in the signaling pathway is supported by our in vitro experiments showing that culturing neurons in the presence of MAG-expressing cells or a MAG-Fc chimera caused elevated ERK 1/2 and cdk5 activities and increased expression and phosphorylation of neurofilaments. Furthermore, the in vitro models provide a means for further investigation of the molecular mechanisms of MAG-mediated signaling by pharmacological approaches.